Efficient fermentation production method of brevibacitracin S
Through the fermentation method of composite inducer and dynamic feeding strategy, the problems of low yield and high cost in the production of gramicidin S are solved, and efficient gramicidin S production is achieved, which has broad prospects for industrial application.
Patent Information
- Application Number
- CN202510655749.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-21
- Publication Date
- 2025-09-16
AI Technical Summary
In the prior art, the production of gramicidin S mainly relies on microbial fermentation, but has problems such as low yield and high production cost, which limits its further application.
A fermentation method using a composite inducer and a dynamic feeding strategy was used. By controlling the pH value and glucose concentration of the fermentation broth in real time and adding inducers in stages, the expression of the gramicidin synthetase gene cluster was activated and the fermentation process was optimized.
The method significantly increases the yield of gramicidin S, reduces the accumulation of fermentation by-products, simplifies the operation process, and has broad prospects for industrial application.
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Figure CN120648768A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of microbial fermentation, and relates to an efficient fermentation production method of Gramicidin S (GS), in particular to a fermentation method for improving the yield of Gramicidin S based on a composite inducer and a dynamic feeding strategy. Background Art
[0002] Gramicidin S (GS) is a cyclic decapeptide antibiotic produced by Bacillus brevis. Its molecular structure contains four amino acids: leucine (Leu), valine (Val), ornithine (Orn), and phenylalanine (Phe). It has broad-spectrum antibacterial activity and is widely used in medicine and agriculture.
[0003] At present, the production of GS mainly relies on microbial fermentation, but the traditional fermentation process has problems such as low yield and high production cost, which limits its further application. Summary of the Invention
[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a method for the efficient fermentation production of gramicidin S. The method is based on a composite inducer and a dynamic control design, which can effectively increase GS yield and reduce production costs.
[0005] To achieve the above object, the present invention adopts the following technical scheme: a method for efficiently fermenting gramicidin S, wherein the fermentation is performed by Bacillus brevis; during the fermentation process, the pH value of the fermentation broth is maintained at 6.5-7.0, and the concentration of glucose in the fermentation broth is maintained at 5-10 g / L by real-time dynamic feeding; and 0.5-1.2 g / L of L-valine, 0.3-0.8 g / L of a phenylalanine derivative, and 0.1-0.5 g / L of a D-amino acid precursor are added as inducers, based on the volume of the fermentation broth.
[0006] In some embodiments, the fermentation is first performed by activating the strain and preparing the seed liquid: Bacillus brevis is inoculated into the seed culture medium, and cultured at 35-38°C and 150-250 rpm for 12-18 hours to obtain the seed liquid (OD 600nm=8-12). Fermentation culture is then carried out: the seed liquid is inoculated into the fermentation medium at an inoculum size of 5-10% (v / v), and the fermentation culture is carried out at 35-38°C and 150-250rpm, with the ventilation volume maintained at 1.0VVM. During the fermentation process, a dynamic feeding strategy is adopted to add feed medium according to the changes in glucose concentration and pH value in the fermentation broth. A composite inducer system is added to the fermentation broth, including a composition containing L-valine (0.5-1.2g / L), a phenylalanine derivative (0.3-0.8g / L) and a D-amino acid precursor (0.1-0.5g / L) to activate the expression of the gramicidin synthetase gene cluster. It should be noted that different inducer combinations, different intervention times, and different amounts of access all produce different results. When the GS concentration in the fermentation broth reaches the maximum, the fermentation is terminated, the bacteria are centrifuged, and the GS is extracted and purified.
[0007] Furthermore, the dynamic feeding strategy is:
[0008] When the glucose concentration in the fermentation broth is lower than 5g / L, start adding feed medium. The addition speed is adjusted according to the pH value of the fermentation broth:
[0009] When the pH value of the fermentation broth is lower than 6.5, reduce the feeding rate to 0.5-1.0 mL / min;
[0010] When the pH value of the fermentation broth is higher than 7.0, increase the feeding rate to 2.0-3.0 mL / min;
[0011] The glucose concentration in the fermentation broth was controlled to be maintained at 5-10 g / L and the pH value was maintained at 6.5-7.0.
[0012] In some preferred embodiments, during the real-time dynamic feeding process, the feeding rate is regulated using a PID control algorithm with a control accuracy of ±0.1 pH unit.
[0013] Furthermore, the feeding strategy of the composite inducer is:
[0014] Phased dynamic addition strategy: Inducers were added in three gradient additions during the fermentation cycle. The first addition was at the end of the logarithmic growth phase of the bacteria (OD600 ≥ 10), and 0.5-1.2 g / L of L-valine, 0.3-0.8 g / L of phenylalanine derivatives, and 0.1-0.5 g / L of D-amino acid precursors were added as 1 part by weight based on the volume of the fermentation broth. The second addition was at the initial stage of product synthesis (pH returned to 6.8-7.0), and the third addition was within 2 hours after the dissolved oxygen inflection point, with 0.5 parts by weight added respectively.
[0015] Furthermore, the fermentation medium comprises the following components:
[0016] Glucose 20-40g / L;
[0017] Yeast powder 10-20g / L;
[0018] Ammonium sulfate 5-10g / L;
[0019] Potassium dihydrogen phosphate 2-5g / L;
[0020] Magnesium sulfate 0.5-1g / L;
[0021] Trace elements 0.1-0.5g / L.
[0022] Furthermore, the feed medium used in the real-time dynamic feeding comprises the following components:
[0023] Glucose 400-600g / L;
[0024] Yeast powder 100-200g / L;
[0025] Ammonium sulfate 50-100g / L;
[0026] Potassium dihydrogen phosphate 20-50g / L;
[0027] Magnesium sulfate 5-10g / L;
[0028] Trace elements 1-5g / L.
[0029] Furthermore, the trace elements include FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O, and CuSO4·5H2O, and the mass ratio of each component is (1-3):(0.5-2):(0.1-1):(0.05-0.5).
[0030] In some preferred embodiments, the composite inducer system comprises the following components: 0.8 g / L L-valine, 0.5 g / L 4-fluorophenylalanine, and 0.2 g / L D-phenylglycine.
[0031] The beneficial effects of the present invention are as follows:
[0032] (1) The present invention uses a dynamic feeding strategy to effectively control the glucose concentration and pH value during the fermentation process, providing an optimal environment for bacterial growth and GS synthesis, thereby increasing GS production. The present invention also uses a composite inducer to activate the expression of the gramicidin synthetase gene cluster and proposes the optimal timing and frequency of addition to further increase GS production. The application of the above optimization strategy ultimately increases gramicidin S production by 3-4 times while reducing the accumulation of fermentation by-products.
[0033] (2) The fermentation method provided by the present invention is simple to operate, easy to industrialize and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS
[0034] Figure 1 This is the high performance liquid chromatography (HPLC) spectrum of the fermentation broth of Example 1 of the present invention. DETAILED DESCRIPTION
[0035] The following describes embodiments, features, and aspects of the present invention in detail with reference to the accompanying drawings. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. The terms used in the present invention are for the purpose of describing specific embodiments only and are not intended to limit the present invention.
[0036] Unless otherwise specified, the materials and reagents used in the following examples are all common commercial products and can be purchased on the market.
[0037] Example 1
[0038] 1. Bacteria activation and seed liquid preparation: Inoculate Bacillus brevis into the seed culture medium and culture at 35-38°C and 150-250 rpm for 12-18 hours to obtain the seed liquid. Control the OD value of the seed liquid. 600nm At 8-12.
[0039] 2. Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculum rate of 5-10% (v / v), and carry out fermentation culture at 35-38°C and 150-250 rpm. The ventilation volume during the fermentation process is maintained at 1.0 VVM.
[0040] 3. Dynamic feeding: During the fermentation process, a dynamic feeding strategy was adopted to add feed medium according to the changes in glucose concentration and pH value in the fermentation broth. The feed medium contained 500 g / L glucose, 150 g / L yeast extract, 75 g / L ammonium sulfate, 40 g / L potassium dihydrogen phosphate, 7.5 g / L magnesium sulfate and 3 g / L trace elements.
[0041] 4. Compound inducer induction, dynamic induction: first addition: OD 600nm When the pH reaches 10 (the end of the logarithmic growth phase of Bacillus brevis), add 0.8 g / L of L-valine, 0.5 g / L of 4-fluorophenylalanine and 0.2 g / L of D-phenylglycine (based on the volume of the fermentation broth); second addition: when the pH returns to 7.0 (the initial stage of product synthesis), add 50% (w / w) of the total amount of the first inducer; third addition: after a sudden drop of dissolved oxygen by 30%, add the remaining amount (50% of the total amount of the first inducer) within 2 hours.
[0042] 5. Fermentation termination and product extraction: When the GS concentration in the fermentation broth reaches 4.2 g / L, the fermentation is terminated, the bacteria are separated, and the GS is extracted and purified. Figure 1 Shown is the HPLC profile of the fermentation broth of this example.
[0043] Example 2
[0044] 1. Bacteria activation and seed liquid preparation: Inoculate Bacillus brevis into the seed culture medium and culture at 35-38°C and 150-250 rpm for 12-18 hours to obtain the seed liquid. Control the OD value of the seed liquid. 600nm At 8-12.
[0045] 2. Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculum rate of 5-10% (v / v), and carry out fermentation culture at 35-38°C and 150-250 rpm. The ventilation volume during the fermentation process is maintained at 1.0 VVM.
[0046] 3. Dynamic feeding: During the fermentation process, a dynamic feeding strategy was adopted to add feed medium according to the changes in glucose concentration and pH value in the fermentation broth. The feed medium contained 500 g / L glucose, 150 g / L yeast extract, 75 g / L ammonium sulfate, 40 g / L potassium dihydrogen phosphate, 7.5 g / L magnesium sulfate and 3 g / L trace elements.
[0047] 4. Fermentation termination and product extraction: When the GS concentration in the fermentation broth reaches 1.0 g / L, the fermentation is terminated, the bacteria are separated, and the GS is extracted and purified.
[0048] Example 3
[0049] 1. Bacteria activation and seed liquid preparation: Inoculate Bacillus brevis into the seed culture medium and culture at 35-38°C and 150-250 rpm for 12-18 hours to obtain the seed liquid. Control the OD value of the seed liquid. 600nm At 8-12.
[0050] 2. Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculum rate of 5-10% (v / v), and carry out fermentation culture at 35-38°C and 150-250 rpm. The ventilation volume during the fermentation process is maintained at 1.0 VVM.
[0051] 3. Dynamic feeding: During the fermentation process, a dynamic feeding strategy was adopted to add feed medium according to the changes in glucose concentration and pH value in the fermentation broth. The feed medium contained 500 g / L glucose, 150 g / L yeast extract, 75 g / L ammonium sulfate, 40 g / L potassium dihydrogen phosphate, 7.5 g / L magnesium sulfate and 3 g / L trace elements.
[0052] 4. Induction with compound inducer, first addition: OD 600nm When the pH reaches 10, add 0.8 g / L of L-valine, 0.5 g / L of 4-chlorophenylalanine and 0.2 g / L of D-tyrosine (based on the volume of the fermentation broth); second addition: when the pH returns to 7.0, add 50% (w / w) of the total amount of the first inducer; third addition: after the dissolved oxygen drops by 30%, add the remaining amount (50% of the total amount of the first inducer) within 2 hours.
[0053] 5. Fermentation termination and product extraction: When the GS concentration in the fermentation broth reaches 3.2 g / L, the fermentation is terminated, the bacteria are separated, and the GS is extracted and purified.
[0054] Example 4
[0055] 1. Bacteria activation and seed liquid preparation: Inoculate Bacillus brevis into the seed culture medium and culture at 35-38°C and 150-250 rpm for 12-18 hours to obtain the seed liquid. Control the OD value of the seed liquid. 600nm At 8-12.
[0056] 2. Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculum rate of 5-10% (v / v), and carry out fermentation culture at 36-38°C and 150-250 rpm. The ventilation volume during the fermentation process is maintained at 1.0 VVM.
[0057] 3. Dynamic feeding: During the fermentation process, a dynamic feeding strategy was adopted to add feed medium according to the changes in glucose concentration and pH value in the fermentation broth. The feed medium contained 500 g / L glucose, 150 g / L yeast extract, 75 g / L ammonium sulfate, 40 g / L potassium dihydrogen phosphate, 7.5 g / L magnesium sulfate and 3 g / L trace elements.
[0058] 4. Induction with composite inducer, adjust the inducer molar ratio to 2:3:1 (L-valine: 4-chlorophenylalanine: D-tyrosine), add the same total amount as in Example 1, first add: OD 600nm Add when the pH reaches 10; second addition: add 50% (w / w) of the total amount of the first inducer when the pH returns to 7.0; third addition: add the remaining amount (50% of the total amount of the first inducer) within 2 hours after the dissolved oxygen drops by 30%.
[0059] 5. Fermentation termination and product extraction: When the GS concentration in the fermentation broth reaches 1.8 g / L, the fermentation is terminated, the bacteria are separated, and the GS is extracted and purified.
[0060] Example 5
[0061] 1. Bacteria activation and seed liquid preparation: Inoculate Bacillus brevis into the seed culture medium and culture at 35-38°C and 150-250 rpm for 12-18 hours to obtain the seed liquid. Control the OD value of the seed liquid. 600nm At 8-12.
[0062] 2. Fermentation culture: Inoculate the seed liquid into the fermentation medium at an inoculum rate of 5-10% (v / v), and carry out fermentation culture at 35-38°C and 150-250 rpm. The ventilation volume during the fermentation process is maintained at 1.0 VVM.
[0063] 3. Dynamic feeding: During the fermentation process, a dynamic feeding strategy was adopted to add feed medium according to the changes in glucose concentration and pH value in the fermentation broth. The feed medium contained 500 g / L glucose, 150 g / L yeast extract, 75 g / L ammonium sulfate, 40 g / L potassium dihydrogen phosphate, 7.5 g / L magnesium sulfate and 3 g / L trace elements.
[0064] 4. Induction with compound inducer, first addition: OD 600nm When the pH reaches 10, add 0.4 g / L of L-valine, 0.1 g / L of 4-chlorophenylalanine and 0.05 g / L of D-tyrosine (based on the volume of the fermentation broth); second addition: when the pH returns to 7.0, add 50% (w / w) of the total amount of the first inducer; third addition: after the dissolved oxygen drops by 30%, add the remaining amount (50% of the total amount of the first inducer) within 2 hours.
[0065] 5. Fermentation termination and product extraction: When the GS concentration in the fermentation broth reaches 2.6 g / L, the fermentation is terminated, the bacteria are separated, and the GS is extracted and purified.
[0066] It should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations are possible. Any modifications, equivalent substitutions, or improvements made without inventive effort shall fall within the scope of protection of the present invention.
Claims
1. A method for efficiently fermenting gramicidin S, characterized in that: The fermentation is carried out by Bacillus brevis; during the fermentation process, the pH value of the fermentation liquid is maintained at 6.5-7.0, and the concentration of glucose in the fermentation liquid is maintained at 5-10 g / L through real-time dynamic feeding; and 0.5-1.2 g / L of L-valine, 0.3-0.8 g / L of a phenylalanine derivative, and 0.1-0.5 g / L of a D-amino acid precursor are added as inducers based on the volume of the fermentation liquid.
2. The method according to claim 1, characterized in that The fermentation method comprises the following steps: inoculating brevis into a seed culture medium, culturing at 35-38° C. and 150-250 rpm for 12-18 hours to prepare a seed liquid, inoculating the seed liquid into a fermentation culture medium at an inoculum amount of 5-10% v / v, and fermenting at 35-38° C. and 150-250 rpm; during the fermentation process, terminating the fermentation when the concentration of gramicidin S reaches a peak, separating the bacterial cells by centrifugation, and purifying the product.
3. The method according to claim 1, characterized in that During the fermentation process, a mixture of L-valine, phenylalanine derivatives, and D-amino acid precursors is added in stages as an inducer: at the end of the logarithmic growth phase of Bacillus brevis, 0.5-1.2 g / L of L-valine, 0.3-0.8 g / L of phenylalanine derivatives, and 0.1-0.5 g / L of D-amino acid precursors are added based on the volume of the fermentation liquid, as 1 part by weight; 0.5 parts by weight of the mixture are respectively added at the initial stage of product synthesis and within 2 hours after the dissolved oxygen inflection point.
4. The method according to claim 1, wherein The culture medium used for fermentation contains the following components: 20-40 g / L glucose, 10-20 g / L yeast powder, 5-10 g / L ammonium sulfate, 2-5 g / L potassium dihydrogen phosphate, 0.5-1 g / L magnesium sulfate, and 0.1-0.5 g / L trace elements.
5. The method according to claim 1, wherein The feed medium used for real-time dynamic feeding contains the following components: glucose 400-600 g / L, yeast powder 100-200 g / L, ammonium sulfate 50-100 g / L, potassium dihydrogen phosphate 20-50 g / L, magnesium sulfate 5-10 g / L, and trace elements 1-5 g / L.
6. The method according to claim 4 or 5, characterized in that The trace elements are a complex of FeSO4·7H2O, MnSO4·H2O, ZnSO4·7H2O, and CuSO4·5H2O, and the mass ratio of each component is (1-3):(0.5-2):(0.1-1):(0.05-0.5).
7. The method according to claim 5, characterized in that The real-time dynamic feeding is used to maintain the pH value of the fermentation broth at 6.5-7.0 and the concentration of glucose in the fermentation broth at 5-10 g / L. Specifically, when the glucose concentration in the fermentation broth is lower than 5 g / L, the feeding program is started, and the feeding rate is adjusted according to the real-time pH value: when the pH is <6.5, the feeding rate is adjusted to 0.5-1.0 mL / min; when the pH is >7.0, the feeding rate is adjusted to 2.0-3.0 mL / min.
8. The method according to claim 7, characterized in that During the real-time dynamic feeding process, the feeding rate is regulated by a PID control algorithm with a control accuracy of ±0.1 pH unit.